This research will be done primarily in the Czech Republic at the Institute of Animal Physiology and Genetics as an extension of NIH grant R37 ND 22681. The mammalian oocyte is the cornerstone for a wide range of promising technologies such as assisted reproduction (human and animal), preservation of genetic diversity, and cell therapies through stem cell technologies. An increasing demand for mature oocytes could be secured by an abundant source of oocytes provided by abattoir ovaries. The current in vitro procedures, however, are not satisfactory as evidenced by the poor developmental potential of the resulting in vitro matured oocytes. This is especially true for the oocytes derived from the numerous small antral follicles. The first Specific Aim of our project is to enhance the developmental competence of these oocytes by establishing a new method that is based on a two-step culture procedure. In this method the oocytes are reversibly inhibited from undergoing maturation by culturing them in the presence of butyrolactone I (BL I), a specific inhibitor of cdk kinases. Following an extended period of culture the oocytes are then matured and fertilized in vitro; the extended culture period appears to foster the acquisition of developmental competence that is dramatically reduced if the oocytes are matured immediately following their collection. The second Specific Aim will determine the suitability of RNA interference, RNAi, to assess gene function in the bovine embryo derived from the two-step culture procedure. The second Specific Aim will determine the suitability of RNA interference, RNAi, to assess gene function in the bovine embryo derived from the two-step culture procedure. In these experiments double-strand RNA (RNAi), will be injected into 1-cell bovine embryos and its ability to promote the selective degradation of the targeted mRNAs (polo-like kinase, p55 Cdc, a critical subunit of the anaphase-promoting complex, and securin, whose destruction is required for sister chromatid separation) will be assessed by RT-PCR. In addition, studies will be conducted to determine the concentration- and time-dependence of this effect, as well as defining the minimum length of RNAi that is required to promote the degradation of the targeted mRNA.